The overall goal of the following experiment is to quantify cellular invasion by the pathogen listeria monocytogenes within the context of irna assays to discover novel cell functions required for bacterial infection. This is achieved by initially performing a cellular irna reverse transfection to inactivate specific host cell functions. As a second step cells are inoculated with listeria monocytogenes, which will differentially infect cells according to the cellular functions that have been affected by the siRNA treatment.
Next infected cells are processed by immunofluorescence in order to label the cellular nuclei. The act in cytoskeleton and the bacterial protein INLC results are obtained that identify infected cells and distinguish them from non-infected cells based on microscopic analysis of the distribution of the INLC protein in individual cells. The main advantage of this technique of existing methods like Gentamycin invasion assays is that it can be used in a FU manner.
So this method may help answer key questions in the cellular microbiology field by identifying novel signaling pathways required for the internalization of eria. Mono is into whole cells 72 hours before cellular infection bring to room temperature a black 384 well microscopy cell culture plate containing 1.6 pico moles IRA in five microliters of RNAs free water in each well to thaw SIR A and to bring down SIR A that could have been deposited in the walls of the wells centrifuge the plate for three minutes at 300 RCF at room temperature. Prepare room temperature DMEM supplemented with 0.4%lip RNAI max.
Do not incubate the transfection medium longer than 20 minutes before adding it to the S-I-R-N-A. Add 25 microliters of the transfection medium to each well of the 384 well plate. Move the plate back and forth to mix the INA with the transfection solution.
Then keep the plate for one hour at room temperature to allow siRNA lip complexes to form wash hela cells from a confluent or sub cofluent cell culture flask once with 10 milliliters of 37 degrees Celsius. Prewarm PBS detach HELOC cells by adding one milliliter 37 degrees Celsius prewarm tripsin to the cell culture flask and incubating for three to five minutes at 37 degrees Celsius. Resus resuspend cells in 10 milliliters of 37 degrees Celsius Prewarm DMEM supplemented with 16%FBS.
After counting cells, prepare a cell suspension of 12, 000 cells per milliliter in DMEM supplemented with 16%FBS add 50 microliters of the cell suspension to each well in the 384 well plate. Move the plate quickly back and forth to distribute the cells, and then let the cells settle down for 10 minutes at room temperature. Seal the plate with param and keep it for 72 hours in a humidified 5%CO2 containing atmosphere at 37 degrees Celsius.
On a pre-warned aluminum block one day prior to infection, take a single colony of L monocytogenes from A BHI auger plate and resus suspend it in five milliliters of liquid BHI medium. In a 15 milliliter polystyrene tube. A highly invasive strain is recommended.
Incubate overnight at 37 degrees Celsius in a shaking device to allow for bacterial growth on the following day. Centrifuge the overnight L monocytogenes culture for two minutes at 10, 600 RCF in a tabletop centrifuge. Discard the supernatant and resuspend the pellet in one milliliter of PBS centrifuge.
Again, discard the supernatant and resuspend the pellet in one milliliter. PBS in this way wash the pellet a total of four times in PBS. After the final PBS wash, read the bacterial optical density at 600 nanometers and estimate the number of bacteria prepare the appropriate L monocytogenes dilution in DMEM supplemented with 1%FBS the use of five times 10 to the fourth bacteria in 30 microliters of medium per well is suggested since the multiplicity of infection is estimated as 25 for 2000 cells retrieved from the incubator.
The 384 well cell culture plate containing the siRNA transfected HELOC cells. Remove the cell culture medium in each well and replace it with 30 microliters of the L monocytogenes containing medium centrifuge. The plate at 200 RCF for five minutes at room temperature to synchronize the infection process.
After that, incubate the plate for one hour in a humidified 5%CO2 containing atmosphere at 37 degrees Celsius on a prewarm aluminum block. Remove the L monocytogenes containing medium from each well and add 30 microliters of prewarm DMEM supplemented with 10%FBS and 40 micrograms per milliliter. Gentamycin to kill extracellular l monocytogenes incubate for four hours in a 5%CO2 containing atmosphere at 37 degrees Celsius on a prewarm metal block.
Once infection of the helo cells with L monocytogenes is complete and extracellular l monocytogenes have been killed by incubation with gentamicin. Add 30 microliters of freshly prepared 8%formaldehyde in PBS to each well. In order to obtain a final formaldehyde concentration of 4%incubate for 15 minutes at room temperature after 15 minutes, remove the fixative and wash the cells three times with 80 microliters of PBS per.
Well keep the cells in a final volume of 80 microliters of PBS per well while the primary antibody solution is being prepared. Remove the PBS from each well and add 10 microliters of the anti INLC primary antibody solution to each. Well incubate for 30 minutes at room temperature.
Prepare the secondary staining solution. Use PBS supplemented with 0.2%saponin to dilute theor 5 46 coupled anti rabid antibody. The DPI solution and PHIN die 6 47 at one to 251 to 1, 501 to 150 respectively.
When the 30 minute incubation is complete, discard the primary antibody solution and wash four times with 40 microliters of PBS per. Well add 10 microliters of the secondary staining solution to each well and incubate for 30 minutes at room temperature after 30 minutes, discard the secondary staining solution and wash four times with 40 microliters PBS per Well leave cells in a final volume of 40 microliters in each well and seal the plate. Subsequently, the plate can be imaged using a 10 x objective mounted on an automated microscope.
Acquire nine images per well in three different channels. Fluorescent labeling of cytoplasmic INLC provides a robust readout for cell infection by L monocytogenes as illustrated in this figure. The central cell in the micrograph is highly infected by the strain P 14 PRFA star as indicated by the arrowheads in the phase contrast image in panel A.This is confirmed in panel B by the blue DPI signal where individual bacteria can be clearly distinguished.
Superposition of the red INLC staining on the DPI staining in panel C depicts how this secreted protein densely accumulates in the cytosol of infected cells and allows an unambiguous detection of the infected host cell morphology in Panel D.Staining of the ACT and cytoskeleton with fluorescent phin superimposed on INLC. Staining provides information concerning the morphology of the completed inoculated, cellular monolayer, and further illustrates how neighboring non-infected cells do not display any INLC labeling. Panel C and D also reveal that INLC is easily detected in the protrusions formed by bacteria caught in the process of cell to cell spread.
Using imaging analysis tools, it is possible to segment cells and to measure the INLC signal in individual infected cells, which can be used to estimate an infection index for a specific experimental condition. The labeling of the nuclei with DPI in panel A and the acton cytoskeleton with PHIN and panel C provide the information required for cellular segmentation. The nuclei are used as reference objects for the identification of individual cells.
In panel B, the DPI signal is blue and the acton signal is red. The cytoplasm is subsequently identified using a spread function from the nuclei that takes into account the cytoskeletal signal signal. Finally, the intensity of the INLC signal can be quantified for each identified cellular object to estimate the number of infected cells.
By setting a threshold for the INLC intensity that is considered as negative. The infection index is calculated as the number of infected cells divided by the total number of cells in the population. Panel E shows the INLC signal and panel F shows the same image as Panel D with superposition of the yellow INLC signal.
This protocol can be coupled to SIR NA screens to investigate the function of large panels of target molecules in the infection of host cells by L monocytogenes. In this example, INLC labeling was examined in HELOC cells inactivated for the cellular receptor. Met by IRNA inactivation of MET leads to a major inhibition of bacterial entry into host cells and very low levels of INLC positive cells in an inoculated cellular monolayer shown in panel A as compared to cells treated with a scrambled irna control shown in panel B.In these images nuclei, acton and INLC are colored blue, red, and yellow respectively using known molecules like met as a standard.
The function of candidate unknown molecules can be investigated with this assay Following this procedure. Other methods like differential, intracellular extracellular bacterial staining can be performed in order to answer additional questions like which specific step of the listeria infection cycle is affected by a given perturbation. After watching this video, you should have a good understanding of how to perform a microscopy basis, say for ster infection that can be upscale for high throughput screens.
